Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)

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dc.contributor.advisor Goldstein , Joseph L . en
dc.creator Yang , Jing en
dc.date.accessioned 2010 -07 -12T17 :23 :26Z en
dc.date.accessioned 2014 -02 -19T22 :01 :21Z
dc.date.available 2010 -07 -12T17 :23 :26Z en
dc.date.available 2014 -02 -19T22 :01 :21Z
dc.date.issued 2009 -06 -19 en
dc.identifier.other 754800058 en
dc.identifier.uri http : / /hdl .handle .net /2152 .5 /271 en
dc.description.abstract Ghrelin is a 28 -amino acid , appetite -stimulating hormone secreted by the food -deprived stomach . Ser -3 of ghrelin is acylated with an eight -carbon fatty acid , octanoate , which is critically required for its endocrine actions . However , the octanoylating enzyme had remained elusive for nearly a decade . By expression cloning , I have identified GOAT (Ghrelin O -Acyltransferase ) , an enzyme belonging to a family of 16 polytopic membrane -bound O -acyltransferases . GOAT activity requires catalytic Asn and His residues , which are conserved through vertebrates . Consistent with its function , GOAT mRNA is largely restricted to stomach and intestine , the major ghrelin -secreting tissues . To further characterize GOAT function biochemically , I have developed a robust in vitro assay using membranes from insect cells infected with baculovirus encoding recombinant mouse GOAT . GOAT -containing membranes catalyze the transfer of [3H]octanoyl from [3H]octanoyl CoA to recombinant proghrelin in vitro . 50 microM palmitoyl CoA is necessary in the assays to prevent the deacylation of [3H]octanoyl CoA by crude membrane preparations . Maximal GOAT activity is observed at pH 7 .0 , and there is no apparent requirement for metals as determined by a lack of inhibition by 1 mM EDTA . The apparent Km for proghrelin is 6 microM and for [3H]octanoyl CoA is 0 .6 microM . The octanoylation reaction strictly depends on the GOAT recognition site comprising three of the four N -terminal amino acids of proghrelin : Gly -1 , Ser -3 , and Phe -4 . A pentapeptide containing only the N -terminal five amino acids of ghrelin is octanoylated by the enzyme . Moreover , I have demonstrated that the activity of GOAT is subjected to end -product inhibition . Together , the insights provided by my research may facilitate the design of useful inhibitors of GOAT . en
dc.format.medium Electronic en
dc.format.mimetype application /pdf en
dc.language.iso en en
dc.subject Acyltransferases en
dc.subject Ghrelin en
dc.subject Octanoic Acids en
dc.title Identification and Biochemical Characterization of Ghrelin O -Acyltransferase (GOAT ) en
dc.type.genre dissertation en
dc.type.material Text en
thesis.degree.name Doctor of Philosophy en
thesis.degree.level Ph .D en
thesis.degree.discipline Cell Regulation en
thesis.degree.grantor Graduate School of Biomedical Sciences en
thesis.degree.department en
dc.format.digitalOrigin born digital en
thesis.date.available 2011 -06 -19 en

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Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT). Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /271 .

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